Elsevier Editorial System(tm) for Saudi Pharmaceutical Journal
Manuscript Draft
Manuscript Number: SPJ-D-19-00715R1
Title: Progesterone loaded thermosensitive hydrogel for vaginal
application: formulation and in vitro comparison with commercial product
Article Type: Original Article
Keywords: progesterone; methylated-β-cyclodextrin; inclusion complexes; in vitro diffusion; drug delivery
Corresponding Author: Dr. Luciano Mengatto, Corresponding Author's Institution: INTEC First Author: Natalia S Velázquez
Order of Authors: Natalia S Velázquez; Ludmila N Turino; Julio A Luna; Luciano Mengatto
Abstract: Progesterone (PGT) is a natural hormone that stimulates and regulates various important functions, such as the preparation of the female body for conception and pregnancy. Due to its low water
solubility, it is administered in a micronized form and/or in vehicles with specific solvents requirements. In order to improve the drug
solubility, inclusion complexes of PGT and β-cyclodextrins were obtained by the freeze-drying method. Two β-cyclodextrins (native and methylated) in two solvents (water and water:ethanol) and different molar ratio of the reagents were the variables tested for the selection of the best condition for the preparation of the complexes. The PGT/randomly methylated-β-cyclodextrin complexes were incorporated into chitosan thermosensitive hydrogels, as an alternative formulation for the vaginal administration of PGT. Neither the micro and macroscopic characteristics of the gels nor the transition time from solution to gel were modified after the complexes incorporation. In addition, chitosan gels with complexes resisted better the degradation in simulated vaginal fluid in comparison to commercial gel (Crinone®). The chitosan gel with inclusion complexes and Crinone® were tested in vitro in a diffusion assay to evaluate the delivery of the hormone and its diffusion through porcine epithelial mucosa obtained from vaginal tissue. Chitosan gel presented sustained diffusion similar to the exhibited by commercial gel. The use of chitosan gels with inclusion complexes based on cyclodextrins would be a viable alternative for vaginal administration of PGT.
Suggested Reviewers: Barbara Luppi [email protected]
Barbara Cometti
[email protected] Sahil Kumar
August 23, 2019
Saleh Alqasoumi, Ph.D. Editor-in-Chief
Saudi Pharmaceutical Journal
We thank you for the revision of our manuscript entitled “Progesterone loaded thermosensitive hydrogel for vaginal application: formulation and in vitro comparison with commercial” by Natalia S. Velázquez, Ludmila N. Turino, Julio A. Luna and Luciano N. Mengatto.
We have analyzed the comments of the reviewers and made corrections (marked in red). New References were added (marked in red). During revision 2 new figures were added: Figure 7 and Figure S3. Figure 7 in the original version of the manuscript is now Figure 8.
We are looking forward to hearing from you shortly. Sincerely yours,
Dr. Luciano Mengatto
P.S.: During the submission process there is no possibility to submit Supplementary Material as a separate file. For this reason I included the Supplementary Material at the end of the Manuscript and submitted them together as a unique file.
Progesterone loaded thermosensitive hydrogel for vaginal application: formulation and in vitro comparison with commercial product
Natalia S. Velázquez, Ludmila N. Turino, Julio A. Luna, Luciano N. Mengatto*
Instituto de Desarrollo Tecnológico para la Industria Química (INTEC), Universidad Nacional del Litoral-Consejo Nacional de Investigaciones Científicas y Técnicas (UNL-CONICET), Centro Científico Tecnológico, Colectora Ruta Nacional 168, Paraje El Pozo,
Santa Fe, Argentina. *
Correspondence: [email protected] Tel.: +54-0342-451-1597 Int.1102
Declarations of interest: none.
Acknowledgments
The authors thank Consejo Nacional de Investigaciones Científicas y Técnicas (Argentina), Consejo Interuniversitario Nacional (Argentina) (Project PDTS N° 453) and Universidad Nacional del Litoral (Argentina) (Project CAI+D 50020150100013 LI) for the financial support.
August 23, 2019
Saleh Alqasoumi, Ph.D. Editor-in-Chief
Saudi Pharmaceutical Journal
We thank you for the revision of our manuscript entitled “Progesterone loaded thermosensitive hydrogel for vaginal application: formulation and in vitro comparison with commercial” by Natalia S. Velázquez, Ludmila N. Turino, Julio A. Luna and Luciano N. Mengatto.
We have analyzed the comments of the reviewers and made corrections (marked in red). New References were added (marked in red). During revision 2 new figures were added: Figure 7 and Figure S3. Figure 7 in the original version of the manuscript is now Figure 8. Please, find enclosed the revised manuscript and our answers to the comments of the reviewers.
We are looking forward to hearing from you shortly. Sincerely yours,
Dr. Luciano Mengatto
2 Answers to Reviewer #2
We acknowledge the revision of our manuscript and the comments made by the Reviewer.
R#2: The chart and its caption should be put together.
A: According to the Guide for Authors of the Journal, Figure captions have to be supplied separately, not attached to the figure.
R#2: Which experiment can explain the sentence 'In addition, chitosan gels with complexes resisted better the degradation in simulated vaginal fluid in comparison to commercial gel (Crinone®)' in ABSTRACT?
A: Stability experiment showed that after 30 minutes of immersion in simulated vaginal fluid, the commercial gel was completely dissolved; while the chitosan gel with inclusion complexes showed greater resistance to degradation at the same time. Please note that the procedure of the stability experiment was mentioned in Subsection 2.6 (Lines 214-217) and the results were discussed in Subsection 3.4 (Lines 404-409) of the original version of the manuscript. Please also check Figure S2 in Supplementary material.
R#2: Why the amount of accumulated PGT in IC solution in 75 hour is less than the amount in 60 hour? The same is true for Crinone® and CHT gel+IC.
A: This is an interesting point, thank you for notice it. As you mentioned, this event is evident for independent assays, that’s means all release experiments from Figure 8 B showed a decrease in accumulated progesterone between 60 and 100 h followed by a new step of progesterone release characterized by a different release rate. As this behaviour is formulation independent, we could postulate that is a membrane effect. In addition, an excess of drug should be applied to the surface of the tissue (infinite dose) to guarantee a negligible depletion. In contrast, when a finite dose of drug is applied, the relationship between the amount of drug permeated and time is not linear. The permeation rate decreases along time. In vitro release studies through porcine epithelial mucosa obtained from vaginal tissue were performed on Franz diffusion cells to evaluate the release of progesterone from the chitosan gel in comparison with the commercial product (Crinone®). Despite the mentioned event, the release of progesterone and its permeation through the tissue was observed. The profiles of the chitosan gel and Crinone® were equivalent.
R#2: What is the advantage of your preparation compared to commercial gel (Crinone®)?
A: The chitosan solution and the β-glycerophosphate disodium salt (GP) solution were mixed to obtain the gel forming mixture. The incorporation of the inclusion complexes (progesterone/cyclodextrin) into the GP solution did not affect the formation of the gel. In the commercial product the direct incorporation of the hydrophobic hormone leads to the formation of drug crystals even with the presence of oil components in the formulation. The chitosan gel was formed in situ and presented a similar in vitro release
3 profile than Crinone®. The chitosan gel showed to be superior in respect to the simplicity of its preparation. In addition, the ability of chitosan to interact with mucus indicates that the residence time of the gel at the vaginal site would be enough to provide localized sustained release of progesterone. This information was discussed in Subsection 3.5 (Lines 467-475) of the original version of the manuscript. However, more information regarding advantages of a chitosan gel for vaginal application was added in the revised version to reinforce the presentation of the results (Subsection 3.5, Lines 475-484).
R#2: Why is the release time 170 h? If the preparation is needed in the vagina for 170 hours, will it affect the life quality of the patient?
A: The in vitro release experiments were performed up to 170 h due to this time ensures that almost all the progesterone (89 %) in the inclusion complexes solution had permeated through the vaginal tissue. For progesterone solution, 100 % of the initial amount of hormone permeated at 60 h. The gels presented a diffusion rate lower than both progesterone and inclusion complexes solutions, because of the presence of the polymeric matrix. The release time (170 h) is not related with the residence time of the preparation at the site of application. In vivo release is expected to be faster than in vitro
performance. This is related with the fact that in the in vitro experiment temperature, agitation and the simulated vaginal fluid are used to emulate the vaginal environment. Many types of bacteria and other microorganisms are present in a healthy vagina. In addition, lysozyme which degrades chitosan can be found. All this components, not present in the in vitro experiments, will contribute to the in vivo degradation of the chitosan gel and to the release of the hormone.
R#2: Will the drug be completely released from the formulation?
A: We assume that a complete release of progesterone will occur. As was mentioned above, in the in vitro experiment conditions to emulate the vaginal environment were used. Many types of bacteria and other microorganisms, and enzymes are present in a healthy vagina. All this components, not present in the in vitro experiments, will contribute to the in vivo degradation of the chitosan gel and to the release of the hormone.
Answers to Reviewer #3
We thank Reviewer #3 for his meticulous revision of our manuscript and helpful suggestions, which contributed to improve the presentation.
R#3: The introductory plan is not effective, so can be given information about the necessity of this formulation in vaginal administration rather than model material. Information and examples of vaginal use of chitosan gel should be given: Tuğcu- Demiröz F., Acartürk F., Erdoğan D., Development of long-acting bioadhesive vaginal gels of oxybutynin: Formulation, in vitro and in vivo evaluations. Int J Pharm. 457 (1): 25-39, 2013. Tuğcu-Demiröz F., Acartürk F., Özkul A., Preparation and Characterization of Bioadhesive Controlled-Release Gels of Cidofovir for Vaginal Delivery. J Biomater Sci Polym Ed. 26 (17): 1237-55, 2015.
4 A: The Introduction section is structure in 5 paragraphs. Paragraphs 1 and 2, presents progesterone, its application in human health by using different formulations and routes of administration with advantages and drawbacks. Paragraph 3, mentions the characteristics of the commercial product Crinone®. Paragraph 4, discusses the problem of low aqueous solubility of progesterone and how it can be solved by the preparation of inclusion complexes with cyclodextrins. Paragraph 5, concludes the Introduction by mentioning the objective of the research. We think that this structure is effective. Nevertheless, following the Reviewer suggestion, we added information about vaginal application of chitosan gels in the revised version (Lines 80-94). New references about this topic were included and are marked in red in the References section.
R#3: Your title and content are incompatible. There is no thermosensitive system in your study.
A: Chitosan, a cationic amino polysaccharide, combined with β-glycerophosphate disodium is an outstanding in situ gel-forming system, which was first reported by Chenite et al. (Chenite A et al., Novel injectable neutral solutions of chitosan form biodegradable gels in situ. Biomaterials 2000; 21: 2155–2161). This system is available at a physiologically acceptable pH and is liquid at room temperature. When the temperature is raised to 37 °C, the physiological temperature, it transforms into a gel. Chitosan and its derivatives have been used to prepare in situ gel-forming systems (a. Ahmadi F et al., Chitosan based hydrogels: characteristics and pharmaceutical applications. Res Pharm Sci 2015; 10: 1–16. b. Liu X et al., A novel thermo-sensitive hydrogel based on thiolated chitosan/hydroxyapatite/beta-glycerophosphate. Carbohyd Polym 2014; 110: 62–69. c. Supper S et al., Thermosensitive chitosan /glycerophosphate-based hydrogel and its derivatives in pharmaceutical and biomedical applications. Expert Opin Drug Deliv 2014; 11: 249–267. d. Zhou H et al., Design and evaluation of chitosan-b-cyclodextrin based thermosensitive hydrogel. Biochem Eng J 2016; 111: 100–107). The thermosensitive system based on chitosan and β-glycerophosphate disodium salt used in the present contribution was characterized and reported in a previous manuscript published by some of the authors (Mengatto LN et al., Application of simultaneous multiple response optimization in the preparation of thermosensitive chitosan/ glycerophosphate hydrogels. Iran Polym J 2016; 25: 897– 906). In that work, the information referring to ability of the solution to form the gel, temperature and time for sol/gel transition was studied. An optimization process based on response surface methodology was carried out in order to develop statistical models that describe the relationship between parameters affecting the sol/gel transition and final properties of the gel. These equations can be used to prepare gel-forming solutions with desired pH, gelling time and residual mass, intended for controlled drug delivery.
R#3: Line 76-82: the aim of the study should be explained in detail and clearly.
A: According to the Reviewer suggestion, we rewrote the aim to render it more clear (Lines 90-95).
R#3: Line 89: information on chitosan MW and degree of deacetylation should be indicated because these values have an effect on the formulation as in the given references: Tuğcu-Demiröz F., Acartürk F., Erdoğan D., Development of long-acting bioadhesive vaginal gels of oxybutynin: Formulation, in vitro and in vivo evaluations. Int J Pharm 457 (1): 25-39, 2013. Tuğcu-Demiröz F., Acartürk F., Özkul A.,
5 Preparation and Characterization of Bioadhesive Controlled-Release Gels of Cidofovir for Vaginal Delivery. J Biomater Sci Polym Ed. 26 (17): 1237-55, 2015.
A: We agree with the Reviewer that MW and DD can influence some final properties of formulations based on chitosan. The study of these influences in the chitosan formulation developed was not the aim of this work. We have expertise in the field of chitosan based formulations and we use a chitosan pharmaceutical degree for the preparation of our systems. Following the Reviewer suggestion, the information concerning to MW and DD was added in the revised version of the manuscript (Lines 102-103).
R#3: Line 102: Describe in more detail the method you use to analyze progesterone with hplc. Is the current method in simulated vaginal fluid?
A: According to these authors, the HPLC methodology is explained in detail. We gave information about equipment, column, oven temperature, mobile phase composition, flow rate, wavelength of detection and procedure for the preparation of standard solutions to obtain the calibration curve. The solvent used for the preparation of standard solutions and samples was clarified in the revised version to answer the specific question made by the Reviewer (Lines 124-127).
R#3: Line 114: explain why the solubility study was done and why water and ethanol-water were chosen.
A: Please note that in the subsection 3.1 Phase solubility studies, we explained the aim of this study and the information obtained from them. This study describes how the increase in cyclodextrin concentration influences drug solubility and are a good starting point to increase knowledge about the interaction between the cyclodextrin and the drug. From this study a researcher can obtain information about the solubility of the complex and the value of the Apparent Stability Constant which indicates the affinity between the reactants. For the preparation of the complexes, using the freeze-drying method, different solvents can be used: water, buffer, organic solvents or a mixture of them. The reason of our choices is: -Water: is the most widely used. -Water:ethanol: according to published works on the subject cosolvents, such as ethanol, can enhance the solubility of hydrophobic drugs and this will lead to enhanced the complexation efficiency (Loftsson, T., Brewster, M.E., 2012. Cyclodextrins as functional excipients: methods to enhance complexation efficiency. J. Pharm. Sci. 101, 3019-3032). Different proportion of water:ethanol or other organic solvents could be used but that was not the objective of our work.
R#3: Line 133: the device used in freeze-drying and its characteristics should be indicated.
A: Following the Reviewer suggestion, the device used in freeze-drying procedure was clarified (Lines 153-154).
R#3: Line 186: viscosity, rheological and textural studies on chitosan gel can be added. Because these studies give important information for drugs to be applied to the vagina. What is the pH of the vaginal formulation that you develop is suitable for vaginal application?
6 A: Following the reviewer suggestion, viscosity and rheological studies were carried out (Lines 218-228). Results are presented in Lines 410-432 of the revised version of the manuscript. Textural studies were not carried out. According to our expertise and knowledge these tests are performed to evaluate the “mouth feel” properties of food, creams or pastes. Since this test is intended to reflect the human perception of texture, the compression cycles mimic the human bites (Steffe, J.F. 1996. Rheological Methods in Food Process Engineering. 2nd Edition, Freeman Press, East Lansing). As was mentioned in the answer to the second comment, we have developed statistical models that describe the relationship between parameters affecting the sol/gel transition and final properties of the gel. These equations can be used to prepare gel-forming solutions with desired pH, gelling time and residual mass, intended for controlled drug delivery (Mengatto LN et al., Application of simultaneous multiple response optimization in the preparation of thermosensitive chitosan/ glycerophosphate hydrogels. Iran Polym J 2016; 25: 897–906). Therefore, we can prepare chitosan gels that fulfil the requirement concerning the pH for vaginal application.
R#3: What will be the amount of gel to be administered and how much progesterone will it carry? Is the dose of progesterone sufficient? Is it compatible with Crinone?
A: Our first attempt was to develop a formulation with the aim to solve problems associated with crystal drug administration, poor retention, irritation and other undesirable effects at the site of application of actually used commercial products to achieve patient compliance. In this course of action, the use of cyclodextrin allowed the hormone to be in a soluble state in the formulation and the chitosan gel showed a comparable progesterone release to the commercial product. Regarding the irritation problems, these effects would be less significant with the chitosan gel due to the formulation has fewer inactive ingredients and the biocompatibility of the polymer was showed in vaginal formulations based on the lack of toxicity and absence of inflammatory reactions. As we discussed in answers to previous comments, mucoadhesion of the polymer was also reported. Features concerning amount of gel to be administered and dose of progesterone necessary to reach successful in vivo
performance are the following steps under study. Optimization of these parameters involves a complete new study and it is under consideration.
R#3: Line 203: This release study is not in vitro. This study ex-vivo should be regulated accordingly. It is unclear how many repeats the release study has taken.
A: We agree with the Reviewer that an assay carried out by using either human or animal vaginal tissue could be considered ex vivo. Nevertheless, we called our study in vitro based on the difference with those carried out in animals i.e. in vivo. According to the definition of IUPAC, in vitro means: in glass, referring to a study in the laboratory usually involving isolated organ, tissue, cell, or biochemical systems (IUPAC. Compendium of Chemical Terminology, 2nd ed. (the "Gold Book"). Compiled by A. D. McNaught and A. Wilkinson. Blackwell Scientific Publications, Oxford (1997). Online version (2019-) created by S. J. Chalk. ISBN 0-9678550-9-8. https://doi.org/10.1351/goldbook). Therefore, we think it is not incorrect to call the experiment in vitro. At the same time, the study was appropriately designed and carried out taking into consideration other similar studies cited in the manuscript (Monteiro Machado, R., de-Oliveira, A., Gaspar, C., Martinez-de-Oliveira, J.,
Palmeira-7 de-Oliveira, R., 2015. Studies and methodologies on vaginal drug permeation. Adv. Drug Del. Rev. 92, 14-26). Please note that the procedure was carefully described in the Subsection 2.7 (Lines 231-249). In addition, we mentioned that each assay was performed in triplicate (Line 249-249).
R#3: Using too many acronyms is confusing.
A: To render the text less confusing, the following acronyms were replaced: IVF, DS, MP, PSD, PM, FTIR, DSC, DLS, SEM and H-NMR.
R#3: Figure titles should be more detailed and informative, especially not enough figure 7.
A: According to the Reviewer suggestion, Figure captions were rewritten to render them more detailed and informative.
R#3: Is the study aimed at controlled release? 175 hours, 7 days can also stay in the vagina? You need to test it. What is the residence time of this gel in the vagina?
A: The in vitro release experiments were performed up to 170 h due to this time ensures that almost all the progesterone (89 %) in the inclusion complexes solution had permeated through the vaginal tissue. For progesterone solution, 100 % of the initial amount of hormone permeated at 60 h. The gels presented a diffusion rate lower than both progesterone and inclusion complexes solutions, because of the presence of the polymeric matrix. The release time (170 h) is not related with the residence time of the preparation at the site of application. In vivo release is expected to be faster than in vitro
performance. This is related with the fact that in the in vitro experiment temperature, agitation and the simulated vaginal fluid are used to emulate the vaginal environment. Many types of bacteria and other microorganisms are present in a healthy vagina. In addition, lysozyme which degrades chitosan can be found. All this components, not present in the in vitro experiments, will contribute to the in vivo degradation of the formulation and to the release of the hormone.
Regarding the residence time, a comparison between the stability in simulated vaginal fluid of the chitosan gel and the commercial formulation (Lines 404-409 and Figure S2) and results from rheological measurements (Lines 423-432) could indicate a greater resistance to degradation under simulated in vivo conditions for our formulation. In addition, the mucoadhesion properties of chitosan that we discussed in previous comments together with the new information introduced about the application of chitosan gels as an intravaginal formulation contributes to indicate that the residence time of the chitosan gel at the vaginal site would be enough to provide localized sustained release of progesterone.
R#3: Crinone is used once a day. Is the formulation you developed sufficient to release in 24 hours?
A: The analysis of the percentage of accumulated progesterone (%) as a function of time in simulated vaginal conditions, indicated equivalence between the chitosan gel and the commercial formulation. This comparison was carried out according to a model independent approach utilizing a difference factor (f1) and a similarity factor (f2)
8 introduced by Moore and Flanner (Moore JW, Flanner HH. Mathematical comparison of dissolution profiles. Pharm Tech. 1996; 20:64–74). As was mentioned in a previous comment, features concerning amount of gel to be administered and dose of progesterone necessary to reach successful in vivo performance are the following steps under study. Optimization of these parameters involves a complete new study and it is under consideration.
R#3: Accumulate instead of cumulative.
A: We do not understand this comment because the word cumulative was not used in the manuscript.
R#3: Line 426: chitosan has nothing to do with thermosensitivity.
A: The formulation developed in this work is thermosensitive. Please note the answer to the second comment made by the Reviewer.
R#3: Experiments and comments on vaginal application should be added.
A: As was mentioned, in vivo performance of the chitosan gel is the following step under study. Based on the information of the successful vaginal application of similar chitosan formulations discussed above, we are optimist that our system will fulfil with the requirements of the hormone therapy. In addition, vaginal irritation and other undesirable effects at the site of application reported for commercial products would be less significant with the chitosan gel. Our support for this consideration is the fact that the formulation has fewer inactive ingredients and the biocompatibility of the polymer was showed in vaginal formulations based on the lack of toxicity and absence of inflammatory reactions. These comments were developed in Lines 475-484.
Progesterone loaded thermosensitive hydrogel for vaginal application: formulation 1
and in vitro comparison with commercial product 2
Abstract 3
Progesterone (PGT) is a natural hormone that stimulates and regulates various important 4
functions, such as the preparation of the female body for conception and pregnancy. Due to 5
its low water solubility, it is administered in a micronized form and/or in vehicles with 6
specific solvents requirements. In order to improve the drug solubility, inclusion complexes 7
of PGT and cyclodextrins were obtained by the freeze-drying method. Two β-8
cyclodextrins (native and methylated) in two solvents (water and water:ethanol) and 9
different molar ratio of the reagents were the variables tested for the selection of the best 10
condition for the preparation of the complexes. The PGT/randomly methylated-β-11
cyclodextrin complexes were incorporated into chitosan thermosensitive hydrogels, as an 12
alternative formulation for the vaginal administration of PGT. Neither the micro and 13
macroscopic characteristics of the gels nor the transition time from solution to gel were 14
modified after the complexes incorporation. In addition, chitosan gels with complexes 15
resisted better the degradation in simulated vaginal fluid in comparison to commercial gel 16
(Crinone®). The chitosan gel with inclusion complexes and Crinone® were tested in vitro in 17
a diffusion assay to evaluate the delivery of the hormone and its diffusion through porcine 18
epithelial mucosa obtained from vaginal tissue. Chitosan gel presented sustained diffusion 19
similar to the exhibited by commercial gel. The use of chitosan gels with inclusion 20
complexes based on cyclodextrins would be a viable alternative for vaginal administration 21
of PGT. 22
Keywords 23
progesterone; methylated-β-cyclodextrin; inclusion complexes; in vitro diffusion; drug 24
delivery 25
1. Introduction 26
Progesterone (PGT) is a natural steroid hormone involves in the preservation of normal 27
physiology of female reproductive system. In this way, exogenous PGT is used as 28
medication in the prevention of preterm birth, in luteal phase support for in vitro
29
fertilization, in hormone replacement therapy and in endometrial hyperplasia and primary 30
endometrial carcinoma treatment (Scavone et al., 2016). 31
PGT can be administered by different routes: oral, intramuscular and vaginal; having many 32
options for its formulation (Scavone et al., 2016; Almomen et al., 2015; Cometti, 2015). In 33
general, the current commercially available formulations present advantages and 34
drawbacks. For example: capsules for oral administration allow achieving low endometrial 35
concentrations of the drug because of the first pass metabolism by the liver; and 36
intramuscular injections can cause pain and irritation at the injection site reducing the 37
patient's compliance and the therapeutic efficacy. The use of vaginal route can cause 38
vaginal discharge, irritation and mild application site reactions. Nevertheless, it exhibits a 39
preferential absorption, since the drug is transported directly to the uterus (Almomen et al., 40
2015; Cometti, 2015; Choudhury et al., 2011; Lockwood et al., 2014). 41
*Manuscript (WITHOUT Author Information) Click here to view linked References
Crinone® is a white soft gel commercially available in boxes containing 6, 15 or 18 42
applicators. The applicator is designed to deliver a pre-measured dose of the gel directly 43
into the vagina. This gel showed an efficacy comparable to intramuscular formulations, 44
achieving a stable endometrial PGT concentration with low serum levels, while reducing 45
adverse systemic effects (Michnova et al., 2017; Silverberg et al., 2012; Moini et al., 2011; 46
Wang et al., 2015). The carrier vehicle is an oil-in-water emulsion. However, the PGT is 47
only partially soluble in both phases; the majority of the hormone exists as a suspension in 48
micronized scale. In addition, its main components are cross-linked acrylic acid polymers 49
(carbopol and polycarbophil), which accumulate in the tissue generating vaginal irritation, 50
painful sexual intercourse and other side effects (Check, 2009). 51
In pharmaceutical technology, the design and development of novel drug delivery systems 52
aim to increase efficiency of drug delivery and safety in the course of administration and 53
treatment, providing more convenience for the patient. During the optimization of PGT 54
formulations, increasing its aqueous solubility is one of the first problems to be overcome. 55
In some dosage forms, it is necessary the use of oils owing to PGT being practically 56
insoluble in water (7 mg/L or 22.26 μM, at 25 °C). Cyclodextrins (CDs) are cyclic 57
oligosaccharides produced by selective enzymatic synthesis from starch. CDshave a three-58
dimensional structure with a hydrophobic cavity and a hydrophilic exterior, making them 59
useful tools to improve the aqueous solubility of insoluble (or lowly soluble) drugs. Since 60
they are natural products, they have a very low toxic effect and can be used in medicines, 61
food and cosmetics (Messner et al., 2010; Loftsson and Brewster, 2012). In addition to 62
natural CDs, chemically modified CDs are extensively used. In general, CDs consist of 6, 7 63
or 8 glucopyranose units, linked by α-(1-4) bonds, known as α-, β- and γ-CDs, respectively 64
(Messner et al., 2010; Del Valle, 2004). The hydrophobicity of their internal cavity 65
provides them the capacity to act as a host and form stable structures, called inclusion 66
complexes (ICs), with other molecules of very diverse nature (guest). These complexes 67
exhibit new physicochemical characteristics, since bioavailability, water solubility, stability 68
in light presence, heat or oxidation conditions of the included drug are improved, at the 69
same time that unwanted side effects may decrease (Chaudhary and Patel, 2013; Chordiya 70
and Senthilkumaran, 2012; Sharma and Baldi, 2014). ICs between PGT and natural or 71
modified CDs have been prepared by different methods such as precipitation, freeze-drying 72
and spray-drying, providing remarkable improvement in PGT water-solubility (Scavone et 73
al., 2016; Lahiani-Skiba et al., 2006; Zoppetti et al., 2007 b; Cerchiara et al., 2003; 74
Lockwood et al., 2014). 75
With the advent of in vitro fertilization and other assisted reproductive procedures, vaginal 76
formulations of PGT became again the focus of research and one option to administer PGT 77
through this route is gels. CHT is a biodegradable and biocompatible linear polymer which 78
can be used in pharmaceuticals formulations as in situ thermosensitive hydrogels (Mengatto 79
et al., 2016). In addition, CHT gels have been proposed for vaginal delivery of different 80
active ingredients. This polymer possesses remarkable features such as good mucoahesion 81
which improve the retention of the formulation inside the vagina, antimicrobial attributes, 82
and suitable mechanical, release and penetration enhancer properties (Caramella et al., 83
2015; Tuğcu-Demiröz et al., 2015; Cook and Brown, 2018). CHT gels developed for the 84
vaginal delivery of oxybutynin presented the easiest application in comparison to other 85
formulations and histological studies showed that the drug was absorbed without damaging 86
the tissue due to the polymer has preventative effect against cell damage (Tuğcu-Demiröz 87
et al., 2013). Stability and durability studies performed on a CHT based gel for vaginal 88
application of PGT suggested extended residence time due to mucoadhesion and 89
thermosensitive properties of the polymer (Almomen et al., 2015). The aim of our work 90
was the preparation of gels based on CHT and PGT/randomly methylated-β-cyclodextrin 91
complexes. The CHT gel with ICs and Crinone® commercial product were tested in a 92
release experiment to compare the delivery of the hormone and its diffusion performance 93
through porcine epithelial mucosa obtained from vaginal tissue. Therefore, CHT gels 94
containing ICs were studied as an alternative formulation for vaginal application of PGT. 95
96
2. Materials and methods 97
2.1 Materials 98
PGT (MW = 314.45 g/mol, purity 99.2 %) was acquired in Farmabase (Italy). RAMEβ-CD, 99
(MW = 1291.8 g/mol, Degree of substitution = 12) was purchased from Cyclolab 100
(Hungary). β-CD (MW = 1135 g/mol) was donated (Roquette, France). Crinone®
8 % gel 101
(Merck-Serono) was purchased at a local supplier. CHT (MW = 600000 g/mol, Degree of 102
deacetylation = 75-85 %) was purchased from China Easter Group (China). β-103
glycerophosphate disodium salt (GP) was kindly provided by Surfactan S.A. (Argentina). 104
The water was of Milli-Q quality (Millipore, USA). Ethanol (EtOH), acetic acid and lactic 105
acid were analytical grade (Cicarelli, Argentina). Methanol (MeOH) was HPLC grade 106
(Merck, Germany). Isotonic phosphate buffer saline with EtOH (PBS-EtOH, 80:20 v:v, pH 107
= 7) was prepared by mixing 800 mL of PBS with 200 mL of EtOH. PBS was prepared by 108
dissolving 8 g of NaCl, 0.2 g of KCl, 0.2 g of KH2PO4, 1.44 g of Na2HPO4∙2H2O in 1 L of
109
water. Simulated vaginal fluid (SVF, pH = 4.2) was prepared by dissolving: 3.51 g NaCl, 110
1.4 g KOH, 0.222 g Ca(OH)2, 0.018 g of bovine serum albumin, 2 g of lactic acid, 1 g of
111
acetic acid, 0.16 g of glycerol, 0.4 g of urea and 5 g of glucose in 1 L of water (Marques et 112
al., 2011). Potassium bromide (KBr) and reagents of PBS and SVF solutions were 113
analytical grade (Anedra, Argentina). 114
115
2.2 PGT quantification by HPLC 116
The concentration of PGT was determined by a HPLC system (Prominence Series 20A, 117
Shimadzu). The chromatographic separation was performed using a Zorbax Eclipse XDB-118
C18 column (250 x 4.6 mm, 5 μm pore size) (Agilent). The conditions of analysis were: 119
oven temperature 30 °C, mobile phase MeOH:water (95:5, v:v), flow rate 1 mL/min and the 120
wavelength of detection 254 nm (Helbling et al., 2015). 121
A stock solution of PGT (300 μg/mL) in MeOH was prepared. In order to verify the 122
linearity of the analytical procedure within a concentration range of 1-100 μg/mL of PGT, 123
six concentration levels of standard solutions were prepared in mobile phase and analyzed 124
in triplicate. The calibration curve (Absorbance as a function of PGT concentration) was 125
fitted to a straight line using linear regression analysis. Experimental samples were diluted 126
in mobile phase as necessary. 127
2.3 Phase solubility studies 128
Phase solubility diagrams were obtained following the methodology developed by Higuchi 129
and Connors (1965). A fixed amount of PGT (in excess) and increasing amounts of CD (β-130
CD: 0-2mM or RAMEβ-CD: 0-150mM) were placed in amber glass containers with a 131
specific solvent (water or water:EtOH 50:50, v:v). The containers were stored at 37 °C with 132
orbital shaking (100 rpm) for 1 week. Once the equilibrium was reached, the containers 133
were centrifuged, the supernatants were filtered (0.22 μm) and the concentration of PGT 134
was measured by HPLC. Each experiment was performed in triplicate. 135
The linear portion of the solubility diagrams was fitted to a straight line with slope and 136
intercept. The Apparent 1:1 Stability Constant (K1:1) was calculated according to the
137
Higuchi-Connors equation (Higuchi and Connors, 1965): 138
1 1 lope
o 1- lope Equation 1
139
where So is the intrinsic solubility of PGT in the solvent without CD (y-intercept) and Slope
140
is the slope of the straight line. 141
In addition, the Complexation Efficiency (CE) was calculated (Equation 2). This parameter 142
was proposed by Loftsson and Brewster (2012) and relates the concentration of CD that 143
forms a complex and the concentration of free CD: 144
CE lope
1 - lope Equation 2
145
2.4 Preparation of PGT/CD inclusion complexes 146
PGT/RAMEβ-CD ICs were obtained by the freeze-drying method. First, PGT was 147
dissolved in EtOH and a proportional amount of RAMEβ-CD was dissolved in water 148
(PGT:RAMEβ-CD molar ratios 1:1; 1:5; 1:10 and 1:20). Both solutions were mixed and 149
magnetically stirred for 15 min. Then, the containers were placed in an ultrasonic bath for 5 150
min. The organic solvent was removed under reduced pressure with a rotary evaporator and 151
the solution was centrifuged to remove reagents that did not form a complex. The 152
supernatant was frozen at -80 °C and freeze-dried for 24 hs at 1 mbar pressure in a 153
laboratory freeze dryer (Cryodos -80, Telstar). 154
In order to evaluate the inclusion procedure, efficacy and yield were calculated. 155
The mobile phase used for PGT quantification by HPLC contains MeOH, then it can 156
dissolve the free and the included PGT. In water, only the included PGT will be dissolved 157
(Bouquet et al., 2007). The same amount of sample was dissolved in water and in mobile 158
phase and the solutions were analyzed by HPLC. 159
The Inclusion Efficacy (E%) was calculated according to the following equation:
160
C C
100 Equation 3
161
where Cwater and CMP are the PGT concentrations in the sample dissolved in water and MP, 162
respectively. An E% value near 100 % was considered as a successful interaction and/or
163
formation of complexes between PGT and RAMEβ-CD. 164
The Inclusion Yield (Y%) was calculated according to the following equation:
165
PGTfinal
PGTini ial . 100 Equation 4
where PGTfinal is the mass of PGT recovered at the end of the freeze-dried procedure in the
167
total lyophilized material and PGTinitial is the initial mass of PGT used to prepare the ICs. 168
2.5 Characterization of PGT/CD inclusion complexes 169
Differential scanning calorimetry 170
Approximately 5 mg of the sample were weighed into aluminum capsules and tested on a 171
Mettler DSC821e thermal analyzer (Mettler Toledo). All the assays were carried out under 172
controlled nitrogen atmosphere and with a heating rate of 10 °C/min. The samples were 173
PGT, RAMEβ-CD, and both the physical mixture and the IC in a 1:1 molar ratio. 174
175
Nuclear magnetic resonance 176
Nuclear magnetic resonance spectra were obtained using an AVANCE 300 MHz 177
spectrometer (Bruker). The samples (PGT and the IC in a 1:1 molar ratio) were dissolved in 178
deuterated chloroform (CDCl3) at 25 °C. A chemical shift (δ) of 7.26 ppm for CDCl3 was
179
used as internal reference. 180
The variation of chemical shift (Δδ) of the protons in the PGT due to the inclusion of the 181
hormone into the cavity of the CD was calculated applying the following equation: 182
Δδ δ δ Equation 5
183
where δIC is the proton shift of the PGT in the IC and δFree is the proton shift of the PGT 184
when it is free i.e. not included. 185
186
Dynamic light scattering 187
The hydrodynamic size and size distribution measurements were carried out by dynamic 188
light scattering using a Zetasizer Nano-ZS (Malvern). A sample of the RAMEβ-CD and the 189
IC in a 1:5 molar ratio were dissolved in Milli-Q water and placed in a polystyrene cuvette 190
(dimensions: 1.0 x 1.0 cm). The sample was irradiated with a He-Ne laser (λ 633 nm) at 191
25 ºC. The refractive index was set at 1.33 and the viscosity at 0.8872 cP. The intensity of 192
the scattered light was detected with a backscattering angle of 90 °. 193
194
Scanning electron microscopy 195
Micrographs were obtained by observation with a Phenom ProX Desktop Scanning 196
Electron Microscopy (Thermo Fisher). The observations were made using an acceleration 197
voltage of 15.0 kV. The samples were: PGT, RAMEβ-CD, and both the physical mixture 198
and the IC in a 1:5 molar ratio. 199
200
2.6 CHT gels with inclusion complexes 201
CHT (2% w/w) was dissolved in an acetic acid solution (0.15 M). GP (35 % w/w) and the 202
PGT/RAMEβ-CD ICs were dissolved together in water. Both solutions were mixed and 203
allowed to gel at 37.5 °C (Mengatto et al., 2016). 204
The gels were characterized by Fourier-transform infrared spectroscopy, scanning electron 205
microscopy, stability in SVF and rheological measurements. Infrared spectroscopy studies 206
were performed on a FTIR-8201 PC spectrometer (Shimadzu), in the frequency range of 207
400-4000 cm-1 at a resolution of 8 cm-1 and 40 scans per spectrum. A weighed amount of 208
sample was blended with KBr and compressed to obtain disks. The concentration of each 209
sample in the disk was approximately 1 % in order to obtain more defined spectra. 210
Microscopic observations were carried out with a Phenom ProX Desktop Scanning 211
Electron Microscopy (Thermo Fisher). The samples were frozen and freeze-dried before 212
observations. 213
The stability of the CHT gel with ICs and the commercial gel (Crinone®) in SVF (pH = 4.2) 214
was determined. A weighed amount of the gels was placed in baskets-type containers that 215
were submerged in 20 mL of SVF and were maintained at 37 ºC. At different times the 216
baskets with the gels were gently removed and weighed. 217
Rheological measurements were performed on a rheometer (Haake RheoStress RS80, 218
Haake Instrument Inc.) with parallel plates (35 mm diameter, 2 mm gap). The temperature 219
of the lower plate was maintained at 37 °C with a circulating bath water. Sand paper in the 220
upper plate was used to eliminate slippage (Olivares et al., 2012). The linear viscoelastic 221
region was determined by performing strain sweep tests from 0.01 to 0.1 at 10 Hz. 222
Frequency sweep tests were performed from 0.1 to 10 Hz at strain amplitude of 0.03 (strain 223
deformation within the linear viscoelastic region). The dynamic rheological data obtained 224
included the 2 components of complex shear modulus (G*): the storage modulus (G´) and 225
the loss modulus (G´´), and the complex viscosity (|η*| |G*|/ω, ω frequency of 226
oscillation). The samples were CHT gel+IC and Crinone® freshly prepared and after 15 min 227
of immersion in SVF (37 °C). Each experiment was performed in triplicate. 228
229
2.7 In vitro release experiments 230
Release experiments were performed using a vertical Franz diffusion cell (PermeGear Inc., 231
USA), with a diffusion area of 1.77 cm2 and 12.0 mL of receptor compartment volume. 232
Porcine vaginal tissue, obtained from females between 5 and 6 months of age, was donated 233
by a local slaughterhouse (Figan, Santa Fe, Argentina). Immediately after the animals were 234
sacrificed, the vaginal tissue was removed and placed in PBS until it arrived at the 235
laboratory. 236
The epithelial mucosa was carefully separated, fractionated and stored at -80 ºC. The day 237
before the experiment, a sample of the tissue was thawed and stabilized in buffer solution. 238
Subsequently, it was placed between both compartments with its luminal face towards the 239
donor compartment. The delivery systems tested in the donor compartment were: PGT 240
solution, ICs solution, CHT gel+ICs and commercial gel. The solutions were prepared in 241
SVF:EtOH 80:20 (v:v). In the case of the gels, 0.25 mL of SVF were placed over them to 242
mimic the vaginal environment conditions and prevent gel dryness. The donor compartment 243
was covered to avoid evaporation. The receptor compartment was continuously stirred and 244
maintained at 37.5 °C. EtOH was used to ensure the solubility of the hormone (Monteiro 245
Machado et al., 2015). The initial concentration of PGT in the four systems was the same. 246
amples of 200 μL were withdrawn at regular intervals of time and replaced with fresh 247
medium. The amount of PGT was quantified by HPLC. Each assay was performed in 248
triplicate. 249
The percentage of accumulated PGT (%) was represented as a function of time (t). 250
The permeation profiles were compared using the difference (f1) and similarity (f2) factors
251
(Cascone, 2017). These factors were calculated by the following equations: 252 253 f1 n t 1 Rt n t 1 . 100 Equation 6 254 255 f2 50 . log 1 nt 1 2 -0,5 . 100 Equation 7 256
where n is the number of samples, Rt and Tt are the percentages of drug released from the 257
reference product and the system to be evaluated, respectively, for each time t. Two profiles 258
were considered equivalent when the value of f1 is less than 15 and f2 is in the range 50-100
259 (Cascone, 2017). 260 261 2.8 Statistical analysis 262
ANOVA and Fisher test were used to compare two or more means, respectively. 263
264
3. Results and discussion 265
3.1 Phase solubility studies 266
Solubility diagrams for the β-CD and the RAMEβ-CD are presented in Figure 1 A and B, 267
respectively. Although the study do not verify the formation of the ICs, they describe how 268
the drug solubility increases when CD concentration increases (Messner et al., 2010), 269
providing valuable information about their interaction. Solubility diagrams of the PGT with 270
β-CD in water (Fig. 1 A) showed a behavior of the Bs type, according to the classification
271
of Higuchi and Connors (1965). This suggests the formation of complexes of limited 272
solubility in water (Uekama et al., 1982; Lahiani-Skiba et al., 2006). Solubility diagrams of 273
the PGT with RAMEβ-CD (Fig. 1 B) presented typical curves of the AL type. The diagrams
274
showed that the PGT solubility increases linearly with the concentration of RAMEβ-CD 275
throughout the range of CD concentrations studied, due to the formation of soluble 276
complexes. Lahiani-Skiba et al. (2006) evaluated the behavior of PGT with a trimethylated 277
β-CD and Luppi et al. (2005) with a dimethylated β-CD. These authors reported AL type
278
profiles. The PGT solubility in the absence of RAMEβ-CD (So) was greater in the solution
279
with EtOH in comparison to water. The addition of an organic cosolvent renders the 280
mixture more favorable for the dissolution of a hydrophobic solute such as PGT (Loftsson 281
and Brewster, 2012). 282
Table 1 shows the values of So (y-intercept), K1:1 and CE, calculated with Equation 1 and 2,
283
respectively. K1:1 value for the RAMEβ-CD in water indicated a strong affinity of the CD
284
for the PGT (Luppi et al., 2005; Lahiani-Skiba et al., 2006; Ma et al., 2011). This behavior 285
is due to the high hydrophobicity of the PGT (partition coefficient in an octanol-water 286
system: Poct = 7410; Tomida et al., 1978). Also, the K1:1 obtained in water (74526.09 M-1)
287
was higher than that obtained for the mixture water:EtOH (27.13 M-1). As the organic 288
portion of the medium increases, the apparent constant decreases due to a reduction of the 289
polarity of the medium (Loftsson and Brewster, 2012). 290
The value of the constant for the β-CD (46256.79 M-1
) indicated that the CD is also very 291
effective in forming stable complexes. The difference in the PGT interaction with natural 292
CD and its methylated derivative could be explained taking into account the differences in 293
aqueous solubility (Popielec and Loftsson, 2017). In the RAMEβ-CD, the methyl groups, 294
increase the hydrophobicity of the cavity and facilitate drug binding (Cirri et al., 2005). 295
The estimation of K1:1 is affected by the value of the solubility. Therefore, Loftsson and
296
Brewster (2012) proposed the calculation of CE. This parameter is independent of the 297
solubility and it is more suitable for the determination of the solubilizing effect of CDs. The 298
CE values agreed with typical values reported for aqueous media (CEaverage0.3) (Loftsson
299
and Brewster, 2012). The CE obtained for the CDs in water (β-CD = 0.489 and RAMEβ-300
CD = 0.433) were slightly higher (p < 0.05) than that obtained for the mixture water:EtOH 301
(0.362). Therefore, in water, 1 of each 3 CD molecules is assumed to form a water soluble 302
complex. For RAMEβ-CD in water:EtOH, 1 of each 4 CD molecules formed a water 303
soluble complex (Loftsson et al., 2007). Both CDs can form stable complex with PGT. 304
Nevertheless, RAMEβ-CD was selected for the preparation of the ICs based on its excellent 305
aqueous solubility and due to the obtained complexes also being soluble. 306
307
3.2 Preparation of PGT/CD inclusion complexes 308
The preparation of the complexes was carried out using water and water:EtOH (50:50, v:v) 309
as solvents. Although most of the studies report 1:1 or 1:2 molar ratios (PGT:CD), 310
experiments with higher ratios were done in order to evaluate their effect on the ICs 311
formation. Inclusion Efficacy (E%) and Inclusion Yield (Y%) were calculated with
312
Equations 3 and 4, respectively. In general, these parameters are not reported in the 313
bibliography; however, they are very useful to evaluate the inclusion method. The ICs 314
prepared in water presented E% values greater than 90 %, but Y% values were lower than 40
315
%. For this reason, water:EtOH was the solvent selected for the ICs preparation throughout 316
the work. 317
In Table 2, E% and Y% for ICs prepared in water:EtOH are presented. E% was greater than
318
95 % when RAMEβ-CD concentration increased up to a molar ratio of PGT:RAMEβ-CD 319
1:10. Any further increase in CD concentration (1:20) decreased E%,probably due to CD
320
precipitation (Frömming and Szejtli, 1993 a; Frömming and Szejtli, 1993 b). Regarding 321
Y%, this value was in the range of 77-91 % for molar ratios equal or higher than 1:5. For the
322
molar ratio 1:1, Y% was the lowest (p < 0.05). When higher CD concentrations are used, the
323
CD + PGT ↔ IC equilibrium is displaced to the complexes formation. In addition, other 324
structures between the PGT and the RAMEβ-CD, such as non-inclusion complexes or 325
aggregates can coexist with the ICs (Shakalisava and Regan, 2006; Loftsson et al., 2007). 326
Also, there is an increase in variability with an increase in the concentration of CD. The 327
molar ratio 1:5 was selected for the preparation of ICs for the in vitro release experiments 328
due to E% and Y% presented the best values with the less CD concentration.
329 330
3.3 Characterization of PGT/CD inclusion complexes 331
In order to verify the formation of the ICs, thermal analysis, proton nuclear magnetic 332
resonance, dynamic light scattering and scanning electron microscopy experiments were 333
carried out. The thermal behavior of PGT, RAMEβ-CD, and both the physical mixture and 334
the lyophilized IC in a 1:1 molar ratio was studied (Fig. 2). PGT thermogram showed the 335
characteristic melting peak of the drug at 130.9 °C (Lahiani-Skiba et al., 2006; Zoppetti et 336
al., 2007 a; Li et al., 2018). In the physical mixture thermogram, a shift of the PGT 337
endothermic peak to a slightly lower temperature (127.9 °C) was observed. According to 338
Lahiani-Skiba et al. (2006), the explanation could be the existence of a very weak 339
interaction at high temperatures between the PGT and the CD. In the IC thermogram, the 340
absence of the melting peak of the PGT supported the formation of the complex (Lahiani-341
Skiba et al., 2006; Cerchiara et al., 2003). RAMEβ-CD did not produce any peaks of 342
interest in the studied temperature range. 343
The chemical structure with numbering of protons for each molecule is shown in Figure S1. 344
The nuclear magnetic resonance spectra obtained for the PGT and the IC are presented in 345
Figure 3. Inner protons of the RAMEβ-CD (H3 and H5), and protons H4, H18, H19 and 346
H21 of the PGT (Figure S1) are the most affected during inclusion of the drug into the 347
cavity of the CD (Frömming and Szejtli, 1993 a; Salústio et al., 2009; Zoppetti, 2011). A 348
well-resolved nuclear magnetic resonance spectrum for the RAMEβ-CD was not obtained, 349
possibly due to the presence of residual β-CD content. As a result, only some of the signals 350
could be identified unambiguously. Signals of H3, H5 and H6 produced wide peaks caused 351
by overlapped signals. For this reason, IC formation between PGT and RAMEβ-CD was 352
studied only on the basis of the chemical changes of the drug (Jablan et al., 2011; García et 353
al., 2014).The variation of chemical shift (Δδ) of the protons in the PGT due to the 354
inclusion was calculated (Table 3). The greatest change was observed in H4, indicating that 355
A ring may be the part of PGT involved in IC formation (Uekama et al., 1982). 356
IC and RAMEβ-CD were characterized by dynamic light scattering. Hydrodynamic 357
diameter based on number (Dn) and polydispersity index (PDI) of the samples were 358
obtained. One population was observed in the RAMEβ-CD sample, corresponding to 359
monomeric CD (Dn: 1.1±0.3 nm) with PDI value of 0.567±0.014, which would indicate the 360
presence of some CDs aggregates. The sample with the complexes also displays one 361
population (Dn: 142.8±6.8 nm, PDI: 0.199±0.011) that presented a larger size than the 362
monomeric CD. CDs and ICs can form aggregates, but in most cases they are small 363
(Loftsson et al., 2007). In our case, the aggregates of the PGT/RAMEβ-CD ICs did not 364
affect the optical properties of the solutions since they remained transparent. 365
Electron micrographs of PGT, RAMEβ-CD, physical mixture and IC were obtained (Fig. 366
4). PGT (A) presented a form of irregular granules and RAMEβ-CD (B) displayed hollow 367
spherical particles and porous fragments. The physical mixture (C) showed a mixture of 368
PTG and RAMEβ-CD structures. IC (D) morphology, by contrast, was observed like 369
plates/plane structures with regular edges. The comparison of the images reveals that the 370
ICs are structurally distinct from the CD and the physical mixture, which supports the PGT 371
inclusion into the RAMEβ-CD cavity. 372
373
3.4 CHT gels with inclusion complexes 374
Figure 5 shows infrared spectra of CHT gel, IC, CHT gel+IC, Crinone® and PGT. CHT 375
gel+IC spectrum presented the characteristic peaks of their constituent components, with 376
some variations. The band at 3400 cm-1 corresponding to the superposition of the OH and -377
NH stretching vibrations shifted towards 3465 cm-1. These groups are involved in the 378
formation of inter and/or intramolecular hydrogen bonds (Islam et al., 2013), therefore the 379
IC could present this type of interaction with the CHT molecule. The vibration of the -CH 380
and -CH2 groups of the CD in the region between 2800-3000 cm-1 (2920 cm-1) and the peak
381
corresponding to the group -OCH3 (2852 cm-1) were observed in the CHT gel+IC spectrum.
382
The intensity of the band at 1650 cm-1 corresponding to C=O stretching vibration in amide 383
I, changed after the addition of the IC to the gel. Similar changes were present in the peaks 384
at 1458, 1419 and 1325 cm−1 corresponding to –OH and –CH bending and C–O stretching 385
of the CHT molecule. In addition, there were significant changes in the spectral shape from 386
900 to 1250 cm−1. The intensity of the bands at 1080 and 980 cm−1 decreased and a clear 387
peak appeared at 1222 cm-1. The IR spectrum obtained of the PGT agreed with the results 388
reported by other authors (Zoppetti et al., 2007 b; Lahiani-Skiba et al., 2006; Cerchiara et 389
al. 2003; Liu et al., 2007; Li et al., 2018). The peaks corresponding to the stretching of the 390
C-H bond of CH2 and CH3 groups were observed in the region 2950-2850 cm-1. The
391
spectrum also showed the peaks at 1699 and 1662 cm-1 of the stretching of the C=O. In 392
addition, the peak corresponding to the stretching of the C=C bond was noticed at 1614 cm -393
1
.Some of the peaks of the PGT appeared in the spectrum of Crinone® which possesses an 394
overly complex matrix to make thorough analysis by this technique. 395
CHT gel, CHT gel+IC and Crinone®electron micrographs were obtained (Fig. 6). CHT gel 396
(A) presented an open structure characterized by interconnected pores (Goycoolea et al., 397
2011; Ho et al., 2004). On the other hand, the CHT gel+IC (B) presented a microstructure 398
similar to interconnected sheets with bigger pores. The structure of gels did not present 399
important changes with the incorporation of the IC; they showed a good macroscopic 400
consistency and the formation times were not modified in comparison to those of the blank 401
gel. Crinone® (C) presented a smooth morphology with visible PGT crystals embedded 402
throughout the material and absence of pores. 403
In order to study the stability of the CHT gel+IC and commercial gel in an acid medium 404
(pH = 4.2) similar to in vivo condition, gels were placed in simulated vaginal fluid (SVF) at 405
37 °C. After 30 minutes, the commercial gel was completely dissolved, while the CHT 406
gel+IC showed greater resistance to degradation at the same time (Figure S2). Due to the 407
lack of stability of the commercial gel it was not possible to obtain the variation of the 408
weight over time. 409
The value of the strain amplitude was set at 0.03 (3%) after checked that all measurements 410
were carried out within the linear viscoelastic region, where G* is independent of the strain 411
amplitude (Figure S3). Figure 7 (A and B) shows typical frequency dependence of the 412
storage and loss moduli for CHT gel+IC and Crinone® freshly prepared and after 15 min of 413
immersion into SVF (37 °C). The time of immersion was fixed in 15 min taking into 414
consideration that during stability study the commercial gel was completely dissolved after 415
30 min. All samples showed values of G′ higher than G″ without exhibiting crossing point 416
throughout the frequency range. Both gels can be classified as strong or true gels, because 417
the molecular rearrangements within the network are reduced over the time scales analyzed 418
and G′ is almost independent of the frequency (Rao, 1999). Freshly prepared CHT gel+IC 419
and Crinone® samples show similar viscoelastic characteristics, i.e., the samples presented 420
similar values of G′ and G″. After the immersion into SVF, some differences in the 421
viscoelastic characteristics were noted. While the values of the moduli (G′ and G″) of 422
Crinone® decreased, the values of CHT gel+IC slightly increased. These results suggest 423
that CHT gel+IC better preserves its structure and its strong gel characteristic after being 424
exposed to SVF conditions than Crinone®. Figure 7 (C and D) presents typical curves of 425
complex viscosity as function of frequency. This material function determined under 426
oscillatory shear testing allows the analysis of viscosity behavior of gel-type samples since 427
it is a test that minimally disturbs the material avoiding the gel fracture. It is observed that 428
all samples are shear thinning. However, while Crinone® decreased its viscosity and shear 429
thinning character after the immersion into SVF, the viscosity and shear thinning character 430
of CHT gel+IC increased. These results may indicate that the CHT gel+IC sample with 431
higher viscosity may stay longer inside the vaginal canal. 432
433
3.5 In vitro release experiments 434
Release studies through porcine epithelial mucosa obtained from vaginal tissue were 435
performed on Franz diffusion cells with four different systems in the donor compartment: 436
PGT solution, ICs solution, CHT gel+IC and Crinone®. The accumulated PGT (%) as a 437
function of time (h) was plotted (Fig. 8). The permeation rate of the PGT in solution was 438
much greater than the rate of the other systems (A). 439
For PGT solution, 49 % and 100 % of the initial amount of PGT permeated at 24 h and 60 440
h, respectively. For ICs solution, at 24 h, 15 % of the drug was accumulated in the receptor 441
compartment and at the end of the assay (170 h) it reached 89 % of PGT. The profiles of 442
PGT and ICs solutions were compared according to a model independent approach utilizing 443
a difference factor (f1) and a similarity factor (f2) using Equation 6 and 7, respectively. The 444
profiles were found to be different because f1 was greater than 15 and f2 less than 50 (f1 = 445
59.6 and f2 = 19.9). It was reported that hydrated CD molecules and ICs are able to 446
penetrate into the lipophilic biological barriers with considerable difficulty (Loftsson and 447
Masson, 2001). At the surface of the biological membrane, the drug is released from the IC; 448
therefore, the CD + Drug ↔ IC equilibrium moves to the left as the drug penetrates the 449
membranes (Stella et al., 1999; Shimpi et al., 2005). The partition of the drug from the 450
cavity into the lipophilic barrier could explain the delay in the PGT permeation from ICs 451
solution, with respect to the PGT solution. This result agrees with the consideration that ICs 452
can retard the release of guest molecules, which is a suitable alternative for controlling 453
release. 454
The amount of PGT permeated from the gels increased slowly along time in a sustained 455
manner (B). However, the diffusion rate from the commercial gel was slightly higher. At 24 456
h, 8 % and 5 % of PGT was accumulated from Crinone® and CHT gel+IC, respectively. At 457
the end of the assay, the percentages were 33 % and 29 % for Crinone® and CHT gel+IC, 458
respectively. f1 and f2 values were 12.0 and 69.5, and indicated equivalence between both 459
gels profiles. In addition, the gels presented a diffusion rate lower than the PGT and ICs 460
solutions, as a result of the presence of the polymeric matrix. There is not only an increase 461
in the pathway that the drug has to migrate to reach the surface of the epithelial mucosa but 462
also two partition equilibriums. In the case of CHT gel+IC, a partition from the CD cavity 463
to the gel and then from the gel to the epithelium. For Crinone®, the first step involves the 464
dissolution of crystals, and then PGT is partitioned into the gel and diffuses to reach the 465
epithelium. 466
It is noteworthy, that a CHT solution containing complexes between PGT and RAMEβ-CD 467
can form a gel in situ, which presents a similar release profile than a commercial 468
formulation. The ICs were well incorporated in the GP solution generating a clear mixture. 469
In the commercial gel the direct incorporation of the hydrophobic hormone leads to the 470
formation of drug crystals (Fig. 6 C) even with the presence of oil components in the 471
formulation. Therefore, the CHT gel+IC shown to be superior in respect to the simplicity of 472
preparation. The ability of CHT to interact with mucus indicates that the residence time of 473
the CHT gel+IC at the vaginal site would be enough to provide localized sustained release 474
of PGT. In addition, vaginal insert based on CHT showed better mucoadhesion than inserts 475
based on carbopol which is one of the main components of Crinone® (Darwesh et al., 476
2018). Vaginal irritation and other undesirable effects at the site of application were 477
reported for Crinone®. These effects would be less significant with the CHT gel+IC due to 478
the formulation has fewer inactive ingredients and the biocompatibility of the polymer was 479
showed in vaginal formulations based on the lack of toxicity and absence of inflammatory 480
reactions (Tuğcu-Demiröz et al., 2013; Darwesh et al., 2018; Cook and Brown, 2018). 481
Crinone® is applied as a gel, while the low viscosity of the thermosensitive CHT 482
formulation ensures the capacity of covering the required surface of the tissue resulting in 483
the in situ formation of a gel mucoadhesive layer at the physiological temperature. 484
485
4. Conclusions 486
Inclusion complexes of progesterone and randomly methylated β-cyclodextrin were 487
obtained by the freeze-drying method. These complexes were included into the 488
glycerophosphate solution during the preparation of chitosan thermosensitive hydrogels. 489
The chitosan gel with inclusion complexes and Crinone®,which is a vaginal gel used to 490
supplement progesterone in women who have luteal phase defect, were tested in vitro in a 491
diffusion assay. This experiment was carried out to evaluate the delivery of the hormone 492
and its diffusion through porcine epithelial mucosa obtained from vaginal tissue. Chitosan 493
gel presented sustained diffusion similar to the exhibited by commercial gel. The use of 494
chitosan gels cont
